Developments in industrially important thermostable enzymes: a review

Cellular components of thermophilic organisms (enzymes, proteins and nucleic acids) are also thermostable. Apart from high temperature they are also known to withstand denaturants of extremely acidic and alkaline conditions. Thermostable enzymes are highly specific and thus have considerable potential for many industrial applications. The use of such enzymes in maximising reactions accomplished in the food and paper industry, detergents, drugs, toxic wastes removal and drilling for oil is being studied extensively. The enzymes can be produced from the thermophiles through either optimised fermentation of the microorganisms or cloning of fast-growing mesophiles by recombinant DNA technology. In this review, the source microorganisms and properties of thermostable starch hydrolysing amylases, xylanases, cellulases, chitinases, proteases, lipases and DNA polymerases are discussed. The industrial needs for such specific thermostable enzyme and improvements required to maximize their application in the future are also suggested.     

Expression of xylanase enzymes from thermophilic microorganisms in fungal hosts

Bulk production of xylanases from thermophilic microorganisms is a prerequisite for their use in industrial processes. As effective secretors of gene products, fungal expression systems provide a promising, industrially relevant alternative to bacteria for heterologous enzyme production. We are currently developing the yeast Kluyveromyces lactis and the filamentous fungus Trichoderma reesei for the extracellular production of thermophilic enzymes for the pulp and paper industry. The K. lactis system has been tested with two thermophilic xylanases and secretes gram amounts of largely pure xylanase A from Dictyoglomus thermophilum in chemostat culture. The T. reesei expression system involves the use of the cellobiohydrolase I (CBHI) promoter and gene fusions for the secretion of heterologous thermostable xylanases of both bacterial and fungal origin. We have reconstructed the AT-rich xynB gene of Dictyoglomus thermophilum according to Trichoderma codon preferences and demonstrated a dramatic increase in expression. A heterologous fungal gene, Humicola grisea xyn2, could be expressed without codon modification. Initial amounts of the XYN2 protein were of a gram per liter range in shake-flask cultivations, and the gene product was correctly processed by the heterologous host. Comparison of the expression of three thermophilic heterologous microbial xylanases in T. reesei demonstrates the need for addressing each case individually.     

Xylanases from marine microorganisms: A brief overview on scope,sources, features and potential applications

Global economic growth often leads to depletion of raw materials and generation of greenhouse gases, as industry manufactures goods at ever increasing levels to keep up with the demand. The currently implemented production processes mostly rely on non-renewable resources, they suffer from high energy consumption, and generate waste that often has a negative environmental impact. Eco-friendly production methods are therefore intensely searched for. Among them, enzyme-based processes are appealing, because of their high substrate and reaction specificity and the relatively mild operation conditions required by these catalysts. In addition, renewable raw materials that allow sustainable production processes are also widely explored. Marine xylanases, which catalyze the hydrolysis of xylan, the major component of lignocellulose, are promising biocatalysts. Since they are produced by microorganisms that thrive in a wide variety of environmental conditions, the enzymes may be active at widely different ranges of pH, temperature, and salt concentrations. These properties are important for their successful application in various industrial processes, such as production of bioethanol, bleaching of paper and pulp, and in the food and feed sector. The present work gives a brief overview of marine sources of xylanases, their classification and features, and of the potential applications of these marine enzymes, especially in sustainable processes in the scope of circular economy.     

Extremophiles as a source of novel enzymes for industrial application

Extremophilic microorganisms are adapted to survive in ecological niches such as at high temperatures, extremes of pH, high salt concentrations and high pressure. These microorganisms produce unique biocatalysts that function under extreme conditions comparable to those prevailing in various industrial processes. Some of the enzymes from extremophiles have already been purified and their genes successfully cloned in mesophilic hosts. In this review we will briefly discuss the biotechnological significance of extreme thermophilic (optimal growth 70–80 °C) and hyperthermophilic (optimal growth 85–100 °C) archaea and bacteria. In particular, we will focus on selected extracellular-polymer-degrading enzymes, such as amylases, pullulanases, cyclodextrin glycosyltransferases, cellulases, xylanases, chitinases, proteinases and other enzymes such as esterases, glucose isomerases, alcohol dehydrogenases and DNA-modifying enzymes with potential use in food, chemical and pharmaceutical industries and in environmental biotechnology. Received: 14 August 1998 / Received revision: 17 February 1999 / Accepted: 19 February 1999     

Enzymology of the thermophilic ascomycetous fungus Thermoascus aurantiacus

Different strains of the thermophilic ascomycetous fungus Thermoascus aurantiacus have been reported in the literature to produce high levels of a variety of industrial interest enzymes (i.e. amylases, cellulases, pectinases and xylanases), which have been shown to be remarkably stable over a wide range of temperatures and appear to have tremendous commercial potential. Most studies on enzyme production by T. aurantiacus are carried out in chemically defined liquid medium, under conditions suitable for induction of a particular enzyme. A few studies have investigated the production of some enzymes by T. aurantiacus by solid-state fermentation, using lignocellulosic materials. The present review focuses on the enzymes produced by T. aurantiacus, their main kinetic parameters, and the effect of different culture conditions on production and enzyme activity. It also provides a view of the possible applications of T. aurantiacus enzymes, considering that this thermophilic fungus could comprise a potential source of thermostable enzymes.     

Industrial relevance of thermophilic Archaea

The dramatic increase of newly isolated extremophilic microorganisms, analysis of their genomes and investigations of their enzymes by academic and industrial laboratories demonstrate the great potential of extremophiles in industrial (white) biotechnology. Enzymes derived from extremophiles (extremozymes) are superior to the traditional catalysts because they can perform industrial processes even under harsh conditions, under which conventional proteins are completely denatured. In particular, enzymes from thermophilic and hyperthermophilic Archaea have industrial relevance. Despite intensive investigations, our knowledge of the structure-function relationships of their enzymes is still limited. Information concerning the molecular properties of their enzymes and genes has to be obtained to be able to understand the mechanisms that are responsible for catalytic activity and stability at the boiling point of water.     

Microbial production,characterization and applications of feruloyl esterases

Feruloyl esterases (FAEs) act synergistically with xylanases to hydrolyze ester-linked ferulic (FA) and diferulic (diFA) acid from cell wall material and therefore play a major role in the degradation of plant biomass. The potential applications of these enzymes with reference to agriculture, food and pharmaceutical industries, are discussed in this review. FAE activities produced by different microorganisms are compared for both submerged and solid state fermentations. In addition, their physicochemical properties and molecular biology are presented.     

嗜热菌——工业用酶的新来源

综述了嗜热菌和极端嗜热菌产生的热稳定性的淀粉酶、纤维素酶、环糊精酶、木聚糖酶、几丁质酶、葡萄糖异构酶、蛋白酶等的研究进展及其在食品、化工、环保等方面的应用前景。     

Enzymes from extremophiles

The industrial application of enzymes that can withstand harsh conditions has greatly increased over the past decade. This is mainly a result of the discovery of novel enzymes from extremophilic microorganisms. Recent advances in the study of extremozymes point to the acceleration of this trend. In particular, enzymes from thermophilic organisms have found the most practical commercial use to date because of their overall inherent stability. This has also led to a greater understanding of stability factors involved in adaptation of these enzymes to their unusual environments.     

The Guts of Herbivorous Invertebrates: Promising Sources of Diverse Microorganisms with Unique Hemicellulolytic Systems

Invertebrates including insects are heterotrophic organisms and widely distributed in ecosystems. Due to their superior ability to digest various types of plant biomass taken as foods, some herbivorous invertebrates have attracted a great deal of industrial attention because such organisms include diverse cellulolytic and hemicellulolytic symbionts in their gut. Recent studies have shown that some of gut microorganisms of herbivores possess one or more extracellular fibrolytic enzymes with unique functions, which can be exploited as useful biocatalysts in various bioindustrial fields. Specifically, microbial hemicellulases with favorable biocatalytic activities are expected to be used for the development of excellent animal feed additives, production of prebiotics such as xylo‐ and mannooligosaccharides, and pretreatment of lignocellulosic biomass for the preparation of fermentable sugars. Here, we review our recent studies accomplished on several hemicellulolytic bacteria isolated from the guts of invertebrates and their glycoside hydrolases such as endo‐β‐1,4‐xylanases and endo‐β‐1,4‐mannanases.     

Industrial applications of hyperthermophilic enzymes: a review

Over the past two decades, research scientists have been involved in the investigation of thermophilic and hyperthermophilic microorganisms owing to the unique features of their enzymic systems. Such in-depth investigations are now on their way to mastering the cloning and industrial exploitation of a broad variety of genes encoding enzymes involved in starch hydrolysis, amino acid biosynthesis, protein hydrolysis, etc. In this work, we review the state of the art and future perspectives of industrial applications of enzymes from hyperthermophilic and extreme thermophilic microorganisms, special attention being paid to the biotechnological methods involved in their industrial exploitation.     

A review of biological delignification and detoxification methods for lignocellulosic bioethanol production

Future biorefineries will integrate biomass conversion processes to produce fuels, power, heat and value-added chemicals. Due to its low price and wide distribution, lignocellulosic biomass is expected to play an important role toward this goal. Regarding renewable biofuel production, bioethanol from lignocellulosic feedstocks is considered the most feasible option for fossil fuels replacement since these raw materials do not compete with food or feed crops. In the overall process, lignin, the natural barrier of the lignocellulosic biomass, represents an important limiting factor in biomass digestibility. In order to reduce the recalcitrant structure of lignocellulose, biological pretreatments have been promoted as sustainable and environmentally friendly alternatives to traditional physico-chemical technologies, which are expensive and pollute the environment. These approaches include the use of diverse white-rot fungi and/or ligninolytic enzymes, which disrupt lignin polymers and facilitate the bioconversion of the sugar fraction into ethanol. As there is still no suitable biological pretreatment technology ready to scale up in an industrial context, white-rot fungi and/or ligninolytic enzymes have also been proposed to overcome, in a separated or in situ biodetoxification step, the effect of the inhibitors produced by non-biological pretreatments. The present work reviews the latest studies regarding the application of different microorganisms or enzymes as useful and environmentally friendly delignification and detoxification technologies for lignocellulosic biofuel production. This review also points out the main challenges and possible ways to make these technologies a reality for the bioethanol industry.     

嗜热和嗜碱木聚糖酶研究进展

木聚糖酶是降解半纤维素主要成分木聚糖的关键酶,广泛应用在食品、饲料、制浆造纸、生物脱胶等行业。特别是在造纸工业中,木聚糖酶显示出巨大的应用潜力,已成为国内外研究的热点。纸浆漂白工艺中需要酶在高温碱性条件下发挥作用。目前,主要通过筛选野生型木聚糖酶资源和对现有中性中温木聚糖酶分子改造的方法获得嗜热碱木聚糖酶。文中就嗜热嗜碱木聚糖酶的筛选、嗜热嗜碱机制研究及分子改造进展进行了综述,并对其前景进行了展望。     

Potential role of glycosidase inhibitors in industrial biotechnological applications

The nutrient content of food and animal feed may be improved through new knowledge about enzymatic changes in complex carbohydrates. Enzymatic hydrolysis of complex carbohydrates containing alpha or beta glycosidic bonds is very important in nutrition and in several technological processes. These enzymes are called glycosidases (Enzyme Class 3.2.1) and include amylases, pectinases and xylanases. They are present in many foods such as cereals, but their microbial analogues are often produced and added in many food processes, for instance to improve the shelf-life of bakery products, clear beer, produce glucose, fructose or dextrins, hydrolyse lactose, modify food pectins, or improve processes. However, many plant foods also contain endogenous inhibitors, which reduce the activity of glycosidases, in particular, proteins, peptides, complexing agents and phenolic compounds. The plant proteinaceous inhibitors of glycosidases are in focus in this review whose objective is to report the effect and implications of these inhibitors in industrial processes and applications. These studies will contribute to the optimisation of industrial processes by using modified enzymes not influenced by the natural inhibitors. They will also allow careful selection of raw material and reaction conditions, and future development of new genetic varieties low in inhibitors. These are all new and very promising concepts for the food and feed sector.     

Thermophilic,lignocellulolytic bacteria for ethanol production: current state and perspectives

Lignocellulosic biomass contains a variety of carbohydrates, and their conversion into ethanol by fermentation requires an efficient microbial platform to achieve high yield, productivity, and final titer of ethanol. In recent years, growing attention has been devoted to the development of cellulolytic and saccharolytic thermophilic bacteria for lignocellulosic ethanol production because of their unique properties. First of all, thermophilic bacteria possess unique cellulolytic and hemicellulolytic systems and are considered as potential sources of highly active and thermostable enzymes for efficient biomass hydrolysis. Secondly, thermophilic bacteria ferment a broad range of carbohydrates into ethanol, and some of them display potential for ethanologenic fermentation at high yield. Thirdly, the establishment of the genetic tools for thermophilic bacteria has allowed metabolic engineering, in particular with emphasis on improving ethanol yield, and this facilitates their employment for ethanol production. Finally, different processes for second-generation ethanol production based on thermophilic bacteria have been proposed with the aim to achieve cost-competitive processes. However, thermophilic bacteria exhibit an inherent low tolerance to ethanol and inhibitors in the pretreated biomass, and this is at present the greatest barrier to their industrial application. Further improvement of the properties of thermophilic bacteria, together with the optimization production processes, is equally important for achieving a realistic industrial ethanol production.     

褐藻生物乙醇的研究进展

利用大型褐藻转化生产的第三代燃料乙醇已受到研究者的广泛关注。我国拥有丰富的褐藻资源,具备了褐藻生物乙醇转化的有利条件。为了实现工业化生产,还需要通过筛选分离和基因工程手段获得高效发酵褐藻的优良菌株及优化预处理、发酵条件等。主要介绍了我国褐藻资源概况、预处理方法和微生物发酵褐藻不同组分生产乙醇的研究进展,提出了当前褐藻乙醇转化中存在的问题,展望了褐藻乙醇的发展方向。     

Hydrolytic activities of hydrolase enzymes from halophilic microorganisms

Biomass is normally processed using acidic or basic catalysts, which both have their drawbacks. One suitable alternative is the application of hydrolytic enzymes that can convert biomass into simpler molecules, which can be fermented and processed into biofuel. Hydrolytic enzymes include proteases, lipases, amylases, cellulases, mannanases, chitinases, and xylanases. To discover sources of these enzymes, 19 halophilic strains of microorganisms that are significantly resistant to high salt concentrations were analyzed. The objective of this research was to identify halophilic microorganisms that produce the target enzymes with high activities, and to characterize these enzymes according to their salt tolerances. The results obtained indicated that Pseudolateromonas phenolica, Micrococcus luteus, Pseudoalteromonas peptidolytica, Halomonas socia, Marinobacter maritimus, and Exiguobacterium aurantiacum strain 2 produced the highest protease, lipase, amylase, cellulase, mannanase, chitinase, and xylanase relative activities, respectively. Except for protease from P. phenolica, all the enzymes tested for salt resistance either maintained or increased their activities with increasing NaCl concentration.     

Myceliophthora thermophila syn. Sporotrichum thermophile: a thermophilic mould of biotechnological potential

Myceliophthora thermophila syn. Sporotrichum thermophile is a ubiquitous thermophilic mould with a strong ability to degrade organic matter during optimal growth at 45?°C. Both genome analysis and experimental data have suggested that the mould is capable of hydrolyzing all major polysaccharides found in biomass. The mould is able to secrete a large number of hydrolytic enzymes (cellulases, laccases, xylanases, pectinases, lipases, phytases and some other miscellaneous enzymes) employed in various biotechnological applications. Characterization of the biomass-hydrolyzing activity of wild and recombinant enzymes suggests that this mould is highly efficient in biomass decomposition at both moderate and high temperatures. The native enzymes produced by the mould are more efficient in activity than their mesophilic counterparts beside their low enzyme titers. The mould is able to synthesize various biomolecules, which are used in multifarious applications. Genome sequence data of M. thermophila also supported the physiological data. This review describes the biotechnological potential of thermophilic mould, M. thermophila supported by genomic and experimental evidences.